The present invention relates to polypeptides and polynucleotides encoding same and use thereof in the treatment of medical conditions associated with ischemia.
Angiogenesis is the budding of new blood vessels from pre-existing ones. It occurs in various physiological conditions such as the female reproductive cycle, as well as pathological conditions which include tumors, tissue ischemia and wound healing.
Ischemic heart disease is the leading cause of mortality in many industrialized countries and is responsible for over 500,000 deaths in the US alone each year. Current treatment options include drug therapy, coronary angioplasty and the more invasive coronary artery bypass grafting (CABG).
However, in all these cases, technical issues including the size of the artery involved, lack of appropriate distal vasculature, the complexity of the arterial lesions that cause the occlusion, and the general clinical conditions of the patient frequently prevent revascularization of the ischemic tissues.
A less invasive approach which has recently been developed is therapeutic angiogenesis. This term refers to the introduction of proangiogenic factors aimed at enhancing neovascularization of the ischemic tissue, thus alleviating the ischemia. Two main methods have been utilized in the field of therapeutic angiogenesis; the first is angiogenic gene therapy either in the form of naked DNA or with viral vehicles to deliver various cytokines, the most commonly used being Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF). A more recent method is cell therapy with fully differentiated cells, endothelial progenitor cells or mesenchymal stem cells. Both methods have shown success in animal models and early phase clinical trials.
However, many obstacles still have to be overcome before therapeutic angiogenesis becomes a real clinical alternative for patients suffering from ischemic diseases.
For angiogenic gene therapy, these obstacles include the need for tissue specificity of transgene expression, choice of delivery vehicle, optimization of dose and timing, optimization of route of administration and the potential of adverse events such as edema and tumor development.
Another limitation to the success of angiogenic gene therapy is the lack of maturation of the newly formed vessels and their subsequent regression, thus preventing a significant long-lasting therapeutic effect. This may be because the delivered angiogenic genes are expressed only for a relatively short period of time which does not allow for vessel maturation and recruitment of smooth muscle cells to take place or because multiple angiogenic factors are required for vessel maturation to occur. A possible solution is to use an upstream angiogenic regulator which would activate multiple angiogenic factors simultaneously, thus more closely resembling physiological angiogenesis. Such a factor is hypoxia-inducible factor 1 (HIF-1).
Hypoxia Inducible Factor-1 (HIF-1) is a transcription factor and a master regulator of the response to oxygen deprivation, activating over 40 genes during hypoxia. It is a heterodimeric transcription factor consisting of two subunits; the subunit a is subject to tight regulation by the level of oxygen and is induced during hypoxia, whereas subunit β is constitutively expressed regardless of oxygen tension.
HIF-1α contains two transactivation domains (N-TAD and C-TAD) and an oxygen-dependent degradation domain (ODDD). The protein von-Hippel-Lindau (VHL) interacts with the ODDD of HIF-1α under normoxic conditions and acts as part of an E3-ubiquitin ligase complex, thus sending HIF-1α to proteosomal degradation. In hypoxia, HIF-1α dimerizes with HIF-1β and activates the transcription of its target genes in the nucleus.
Recently, it was shown that the interaction of VHL with HIF-1α is enabled by prolyl hydroxylation of two specific residues within the HIF-1α protein (Epstein, A. C. et al. 2001, Cell 107, 43-54. Masson, N., et al. 2001, Embo J 20, 5197-206).
Three prolyl hydroxylases (PHD 1-3) were found to hydroxylate HIF-1α during normoxia. PHD 1 and 2 hydroxylate at residues 402 and 564 whereas PHD 3 hydroxylates at residue 564 only.
A second mechanism of regulation of HIF-1α was uncovered in 2002 (Lando, D et al, 2002, Science 295, 858-61) and includes the asparaginyl hydroxylation of HIF-1α at residue 803, effected by an asparaginyl hydroxylase, also termed Factor Inhibiting HIF-1 (FIH-1). This hydroxylation prevents the interaction of HIF-1α with the co-factor p300, thus hindering the transcriptional activity of HIF-1α.
Hence, during normoxia two mechanisms of regulation are responsible for the decrease in HIF-1α activity, one concerning its stability via prolyl hydroxylations and the other concerning its transcriptional activation via asparaginyl hydroxylation.
These prolyl and asparaginyl hydroxylases are all dioxygenases which are 2-oxoglutarate and iron dependent and their requirement for cellular oxygen could provide the basis for their activity as oxygen sensors.
Two specific point mutations at residues 402 and 564 were demonstrated to abolish the interaction of HIF-1α with VHL, thus rendering HIF-1α constitutively stable (Masson et al., 2001, Embo J 20, 5197-206). The resultant mutant HIF-1α was as active as hypoxia treatment in driving the expression of an HRE-Luciferase construct. Two mutations at residues 564 and 803 were shown to give HIF-1α full transcriptional activity, similar to that obtained by treatment with the hypoxia mimetic iron chelator 2,2′-Dipyridyl (Lando et al., 2002, Science 295, 858-61).
Transgenic mice overexpressing a mutant hHIF-1α in which residues 401 to 602 are deleted, under the regulation of Keratin 14 promoter showed increased vascularization in the skin (Elson, D. A. et al. 2001, Genes Dev 15, 2520-32). These mice showed up-regulation of mRNA of Glut-1 and VEGF, known targets of HIF-1α. In comparison with VEGF overexpressing mice, these mice had blood vessels which were less leaky and showed greater maturity. This may be explained by the fact that HIF-1α induces the activation of multiple angiogenic factors (i.e. erythropoietin), similar to the physiological angiogenic response, in contrast to VEGF alone. In addition, HIF-1α induces the activation of many isoforms of VEGF and other genes, again more closely resembling the physiological response, which could not be achieved by the administration of a single isoform.
A constitutively active form of HIF-1α was first tested in angiogenic gene therapy in 2000 (Vincent, K. A. et al. 2000, Circulation 102, 2255-61). It contained the DNA binding domain and dimerization domains of HIF-1α attached to the transactivation domain VP16 of the Herpes Simplex Virus (HSV) under the regulation of CMV promoter. The resultant mutant was able to induce HIF target genes in-vitro. When administered locally as naked DNA it caused increased capillary density and blood perfusion in a mouse hindlimb ischemia model. The same construct also showed therapeutic effect when injected IM in a rat MI model (Shyu, K. G. et al, 2002, Cardiovasc Res 54, 576-83).
An adenovirus expressing a constitutively active mutant HIF-1α, with a deletion of residues 401-602, under the regulation of CMV promoter was able to induce angiogenesis in the non-ischemic tissue of the retina (Kelly, B. D. et al, 2003 Circ Res 93, 1074-81).
The two constructs mentioned above bear large deletions of the HIF-1α molecule, and lack one or both of the native HIF-1α transactivation domains (N-TAD and C-TAD), which may result in reduced activation potential and specificity of HIF-1α. In addition, HIF-1α is expressed in these constructs under the regulation of CMV, a versatile and non-specific promoter, which may limit its application due to non-specific expression and potential side-effects.
Various routes of administration have been used to deliver the therapeutic gene to the ischemic region, including intravascular and intramuscular in the case of peripheral ischemia, and intramyocardial, intrapericardial and intracoronary in the case of myocardial ischemia. The intravenous route confers advantages which include easy access without the need for an invasive procedure, technical safety and low cost as well as accessibility to a large patient population. However, despite its obvious clinical appeal, the use of this administration route is uncommon due to systemic distribution of the vector leading to low transgene expression in the target organ along with unwanted expression in non-target organs resulting in systemic side-effects, which limit the dose that may be administered. This limitation, in turn, tends to restrict the efficacy of the treatment. Unwanted expression of a pro-angiogeneic gene could induce pathological angiogenesis, possibly leading to tumor development and retinopathy, and may therefore be unacceptable. Thus, the ability to direct transgene expression specifically to the ischemic target organ is of outmost importance for systemic administration to be efficacious and safe.
There is thus a widely recognized need for, and it would be highly advantageous to have pro-angiogenic factors together with safe and effective methods of delivering same.